Dendrimer Internalization and Intracellular Trafficking in Living Cells
Dendrimer Internalization and Intracellular Trafficking in Living Cells
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DOI：
10.1021/mp9002464
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发表时间：
2010-05-01
影响因子：
4.9
通讯作者：
Beltram, Fabio
中科院分区：
文献类型：
--
作者：
Albertazzi, Lorenzo;Serresi, Michela;Beltram, Fabio
树枝状大分子穿透细胞膜的能力在药物和基因传递方面的应用引起了人们的极大兴趣。最近的研究表明，树枝状大分子能够通过内吞作用进入细胞，但其内化后的细胞内途径仍然存在争议。在这项研究中，我们使用共聚焦荧光显微镜来阐明具有高空间和时间分辨率的PAMAM树状大分子在活的HeLa细胞中的细胞内转运特性。研究了不同化学官能度(中性、阳离子和脂化)、大小(从G2到G6)和表面电荷的大分子及其内化性质与分子结构的关系。毒性和内化数据的讨论，允许识别树枝状大分子最大限度地细胞内摄取，对细胞活力的影响最小。内吞小泡的延时成像和荧光生物标记物的共定位分析表明，树枝状大分子被依赖于笼蛋白的内吞作用和大吞饮作用所内化，并最终被运送到溶酶体隔室。此外，为了治疗的目的，我们分析了树枝状大分子在其他有实际意义的细胞系中的摄取情况。这些测量以及与TAT多肽的直接比较表明，PAMAM树枝状大分子具有与这些广泛使用的细胞穿透肽相似的性质，由于其化学可调整性，可能成为药物和基因输送的有效替代方案。
The ability of dendrimers to cross cell membranes is of much interest for their application in drug and gene delivery. Recent studies demonstrate that dendrimers are capable to enter cells by endocytosis, but the intracellular pathway following their internalization remains controversial. In this study we use confocal fluorescence microscopy to elucidate the intracellular trafficking properties of PAMAM dendrimers with high spatial and temporal resolution in living HeLa cells. Macromolecules of different chemical functionality (neutral, cationic and lipidated), size (from G2 up to G6) and surface charge are investigated and their internalization properties correlated with the molecular structure. Toxicity and internalization data are discussed that allow the identification of dendrimers maximizing intracellular uptake with the minimum effect on cell viability. Time-lapse imaging and colocalization assays with fluorescent biomarkers for endocytic vesicles demonstrate that dendrimers are internalized by both clathrin-dependent endocytosis and macropinocytosis and are eventually delivered to the lysosomal compartment. Moreover we analyzed the uptake of dendrimers in additional cell lines of practical interest for therapeutic purposes. These measurements together with a direct comparison with TAT peptides demonstrate that PAMAM dendrimers possess similar properties to these widely used cell-penetrating peptides and thanks to their chemical tunability may represent a valid alternative for drug and gene delivery.